|
|
Study of factors influencing efficiency of Canabis sativa transformation
Širl, Marek ; Ovesná, Jaroslava (advisor) ; Vondráková, Zuzana (referee)
Hemp (Cannabis sativa L.) is a multi-use crop, able to provide fibre celulose a hurds for industrial treatment seeds for oil preparation biomass for energy conversion and produces secondary metabolites useful for pharmaceutical application. For its resistence to stress ability to accumulate high concentration of heavy metals and low cultivations demands, it can also be used for phytoextractions. Current research is focused on establishment of cultivation protocol, which allows transformation of callus cultures, and their regeneration with high efficiency. In this thesis, several varieties of hemp were transferred to in vitro conditions and were tested for their ability to form callus. The best results were achieved using the hypocotyl segments in a nutrient medium supplemented with 1 mg/L of naphtylacetic acid and one of these two synthetic cytokinins 0,5 mg/L of thidiazuron or 5 mg/L of 6-benzylaminopurine. No significant difference in the use of these two cytokinins were observed. None of the explants on four different test media for regeneration of shoots were able to succesfully regenerate. Transformation of hemp was tested using two different methods. Transformed protoplasts from hemp leafs after agroinfiltration were isolated. This method turn out to be unsuitable for use with hemp due to its...
|
|
|
Transformation of potato with protease inhibitor gene SPI-2
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Vítámvás, Pavel (referee)
The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....
|
|
|
Utilization of biotechnological method in growing of technical hemp
Širl, Marek ; Zelenková, Sylva (advisor) ; Fischer, Lukáš (referee)
Hemp (Cannabis sativa L.) is a multi-use crop, able to provide fibre celulose and hurds for industrial treatment biomass for energy conversion and produces secondary metabolites useful for pharmaceutical application. For its resistence to stress and ability to accumulate high concentration of heavy metals it can be potentialy used for phytoremediation. The aim of current research is the development of new strains, with specialy improved qualities and the enhancement of its applicability. Besides traditional breeding methods, genetic manipulation might be the possible tool. What is currently discussed is finding optimal conditions allowing efective in vitro cultivation as a basic assumption of genetic manipulation. Hemp transformation via Agrobacterium tumefaciens co-cultivation might be the appropriate technic.
|
|
|
Genetically modified plants in practical applications
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Cvrčková, Fatima (referee)
Genetic engineering (GI) of plants is a very current topic, and more and more controversial, since it is becoming an inseparable part of our lives. GI has, among other things, a great potential to help solve the current problem of hunger and malnutrition in certain parts of the world. The goal of this project is to clarify what genetically modified (GM) plants are, to present the possibilities of their practical use, to explain methods of preparation and to consider their advantages and eventual risks. By GM plant we understand a plant whose genetic information has been changed by introducing or removing part of the genetic information (a section of DNA, generally a gene). The most frequently used methods of transformation are the transformation by Agrobacterium tumefaciens and the balistic method. GM plants can be used for production of food or feed in better quality and greater quantity, thanks to an introduction of higher resistance, whether it is to biotic stress (pest, virus or bacterial resistance) or to abiotic stress (drought, salinity, toxic substance in the ground..). In particular, the resistance to abiotic stress is an important issue these days, since through the climate changes some regions suffer more and more from insufficient precipitation and consequent drought. Some of the GM plants grown...
|
|
|
ISOLATION OF TRANSGENIC PLANTS NICOTIANA TABACUM AND SILENE VULGARIS
Kováčová, Viera ; Doškař, Jiří (referee) ; Vyskot,, Boris (advisor)
This project is focused on transformation of Silene vulgaris mediated by Agrobacterium tumefaciens and A. rhizogenes. S. vulgaris is a good model plant to study gynodioecy, an evolutionary step from bisexuality to dioecy. Gynodioecious plants form in some individuals bisexual flowers, while the others possess only female flowers. The aim of this research is do develop a technique to introduce foreign genes into this plant to study its developmental consequences. Using A. rhizogenes we successfuly prepared hairy root cultures, which unfortunately do not form shoot regenerants. We have prepared a protocol to induce plant regenerants from S. vulgaris leaf fragments. The first results do not confirm that A. tumefaciens infected plant regenerants harbor reporter transgenes. We used Nicotiana tabacum as a positive control.
|
|
|
Agrobacterium tumefaciens application
Kováčová, Viera ; Burdychová, Radka (referee) ; Babák, Libor (advisor)
This project focus on biotechnologic applications of bacterium named Agrobacterium tumefaciens, which is capable to mediate genetic transformation not only for plants. Main points of project are divided between description of basic natures and cytology of A. tumafaciens following by overview of usage A. tumefaciens in methods of modifications of diffrent cells. Target of paper about biotechnologic use of A. tumefaciens is confrontation of effectivity of this method of modification with else, summary of positiv benefits of its using for future development of human population and introduction of fundamental transformation methods of modification most important crop plants. Final part of project is brief summation of basic statement from statistics and legislature of genetic modified crops.
|
|
| |
|
|
Analysis of metabolome effects of hop (H. lupulus) transcription factors in heterologous of Petunia hybrida
MORAVCOVÁ, Vendula
Plant transformation is now a key research in plant biology, and also is a unique practical tool in the improvement of varieties of cultivated plants. The aim of this work was to obtain transgenic plants Petunia x hybrida containing transgene nptII (kanamycin resistance gene) using the indirect transformation by Agrobacterium tumefaciens. In terms of in-vivo was grown from seeds experimental material in the form of plant Petunia x hybrida cv. Andrea. For the experiments three different constructs HLWD40 3278-80 and HLbHLH 3577 and HLbHLH 3677 GFP were used. The leaf explants from Petunia plants were prepared. Explants were transferred using tweezers into the crucible containing liquid ? MS and prepared bacteria were added. Thus prepared explants remained in bacteria the next day, when they were transferred to the regenerating solid ground. The explants formed callus gradually (clusters of cells) which regenerate a new plant. Transformation should satisfy the condition that the transformed plants should be fertile. For this reason, an attempt was made crossing the transformed plants were crossed Pap1 1527/2 and Pap1 1572/4 with plants crossed HLWD 40 3278-80. The grown plants were analysed by a PCR reaction. The explants began during the first 2-4 weeks after transfromation to form callus and during the next week to remove the first regenerants. At one exulant fell averaged around 7 regenerants and the explants regenerated by construct HLbHLH 3577 regenerate better than regenerants transformed with construct HLWD 40 3278-80. During the transformation we obtained regenerants. With "tissue" PCR, it was found that 15 plants carried the gene for kanamycin resistance. Transformation efficiency was 3.75%.
|
|
| |
|
| |
guest ::
